The ability of tissues and organs to regenerate throughout life is the result of the presence of tissue-resident stem cells. During homeostasis, dying cells undergo programmed cell death or apoptosis and are replenished by cells descending from stem cells. Stem cell division results in a new stem cell and a cell that differentiates into a mature tissue cell when provided with the proper molecular signals.
After wounding or pathogen infection, however, cells in injured or infected tissue undergo necrosis and release danger-associated or pathogen-associated molecular patterns (DAMPs or PAMPs), respectively. These molecular patterns induce inflammatory processes at the site of injury or infection. During tissue inflammation, acute phase proteins are produced in the liver and are secreted in the blood plasma.
Acute phase proteins including fibrinogen and C-reactive protein activate the complement system, macrophages and cells of the adaptive immune system. Inflammation promotes hemostasis and recruits and activates cells of the innate and adaptive immune system, leading to blood coagulation and the removal of the injured or infected cells. After this, the inflammation stops, the homeostasis is restored and the damaged tissue is repaired by replenishment with stem cell descendants. In case the cell damage extends a certain limit or in case the infection becomes chronic, the plasma levels of acute phase proteins remain elevated and the inflammation becomes exacerbated, which results in excessive deposition of amyloids (plaque formation) and collagen (tissue scarification) leading to a significant loss-of-function of the affected tissue/organ.
Patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia, are treated by hematopoietic stem cell transplantations. In hematopoietic stem cell transplantations, the recipient's immune system is destroyed with radiation or chemotherapy before the transplantation with stem cell transplants from another (allogeneic) individual (the donor). Allogeneic hematopoietic stem cell transplantation to restore hematopoiesis in the recipient remains a dangerous procedure with the graft-versus-host disease as the major complication. Graft-versus-host disease is an inflammatory disease in which cells and components with a pro-inflammatory capability, such as cells of the immune system and acute phase proteins, present in the transplant, attack cells of the recipient. This can occur even if the donor and recipient carry identical major histo-compatibility (MHC) proteins on their surface, because the immune system can still recognize other minor differences between cell surface proteins of the donor and recipient. In addition, the destruction of the recipients' immune system by radiation or chemotherapy prior to the stem cell transplantation results in the accumulation of large numbers of necrotic cells in the bone marrow, which enhances the graft-versus-host response.
Cell necrosis and inflammation is a major driver of tissue damage after insults of the central nervous system and in neurodegenerative diseases. Central nervous system injury including ischemic stroke, traumatic brain injury, and traumatic spinal cord injury represents a major burden to the healthcare system worldwide. Ischemic and traumatic insults of the central nervous system both result in definite chronic disability with an impaired life expectancy. Currently, post-traumatic defects of the central nervous system, such as spinal cord and/or brain injuries, cannot be treated, nor can the underlying histological defects be cured.
The majority of neurodegenerative diseases are sporadic conditions that are characterized by progressive loss of cells in the nervous system. Examples of neurodegenerative diseases are Parkinson disease, Alzheimer's dementia, Huntington's disease, amyotrophic lateral sclerosis and Multiple Sclerosis. The World Health Organization predicts that neurodegenerative disorders will take over cancer to become the second leading cause of death. The available treatments for neurodegenerative diseases aim at improvement of symptoms, pain relief, and increased mobility. However, thus far, the current therapeutics available to treat patients with neurodegenerative diseases, alleviate only the disease symptoms and delay the time to progression to disabling stages.
Stem cell transplantation therapies involving stem cells to restore neurogenesis and provide functional recovery, is an attractive approach to treat nervous system diseases and disorders. Stem cell transplantation therapies to restore vascularization is an attractive approach to treat ischemic diseases and disorders.
Stem cells obtained from embryos, e.g., embryonic stem cells, hold great potential for regenerative medicine (H. Hentze et al., Trends in Biotechnology 25:24-32, 2007), however, they have a number of disadvantages, including the possibility of transplant rejection due to their allogeneic offspring and the possible teratoma formation in case the cells are not securely and quantitatively differentiated prior to transplantation.
Embryonic stem cells need to be cultured and consequently bear the risk of being exposed to xenogenic material or being contaminated with prematurely exhausted cells (F. Mannello and G. A. Tonti, Stem Cells 25:1603-1609, 2007). Due to the limited insight in the biology of embryonic stem cells and their developmental behavior, they currently do not appear to be safe enough for human application.
For the treatment of cancers of the blood or bone marrow, transplantation of allogeneic stem cells derived from the bone marrow or peripheral blood is the most efficient. For the treatment of diseases and disorders of the central nervous system, transplantation of autologous adult stem cells, mainly hematopoietic stem cells and mesenchymal stem cells derived from the bone marrow or peripheral blood, is considered to be the most promising (E. Sykova et al., Cell Transplant 15:675-687, 2006).
The bone marrow stroma harbors heterogeneous populations of multi-potent cells, including hematopoietic stem cells and mesenchymal stem cells capable of self-renewal and differentiation into various cells and tissues of, respectively, the hematopoietic and mesenchymal lineages.
Recent preclinical work investigating the feasibility of stem cell transplantation therapies to treat patients with ischemic stroke, traumatic brain injury, and traumatic spinal cord injury has shown that when applied intrathecally, hematopoietic stem cells and/or mesenchymal stem cells, or descendants thereof, are suggested to infiltrate into the lesioned neural tissue, penetrate glial scar tissue (M. R. Alison, Journal of Pathology 217:141-143, 2009), secrete trophic factors (A. I. Caplan and J. E. Dennis, Journal of Cellular Biochemistry 98:1076-1084, 2006), are capable of differentiation into functional neurons (R. Zeng et al., Spine 36:997-1005, 2011), promote the formation of synaptic connections (F. M. Bareyre, Journal of Neurological Sciences 265:63-72, 2008), and, as a result, participate in the reorganization of the neural network leading to a functional improvement (B. K. Kwon et al., Experimental Neurology 248C:30-44, 2013).
In early stem cell transplantation studies, bone marrow transplants were used, which were manufactured by single or double centrifugation of bone marrow biopsies (WO2007125420). The layer between the erythrocytes and the plasma named the buffy coat was subsequently collected. The buffy coat contains a heterogeneous population of nucleated cells. A significant portion of the nucleated cells are immunity-associated cells with a pro-inflammatory capability and allogeneic transplantation of these cells to restore hematogenesis frequently results in the graft-versus-host disease as stated above. Autologous transplantation of these cells into damaged central nervous system tissue may also lead to adverse or even detrimental effects toward the intended mode of action (K. L. Le Blanc et al., Scandinavian Journal of Immunology 57:11-20, 2003; G. M. Spaggiari et al., Blood 107:1484-1490, 2006; R. A. Adams et al., Journal of Experimental Medicine 204:571-582, 2007; B. Assmus et al., Journal of the American College of Cardiology 55:1385-1394, 2010; and K. D. Beck et al., Brain 133:433-447, 2010). In addition, the chemicals used during the gradient centrifugation step have to be removed from the transplants by introducing additional washing steps to obtain the final transplant product.
Separation of stem cells and progenitor cells from other cell types in tissue biopsies on the basis of their physical characteristics, such as density and sedimentation speed, technically, is very difficult. The limitations of separating stem cells and progenitor cells from other cell types were overcome by the application of monoclonal antibodies that specifically bind to cell surface proteins such as the cluster of differentiation (CD) proteins. Immune adsorption and flow cytometry techniques using the monoclonal antibodies labeled with magnetic beads or fluorescent molecules are currently employed to both positively and negatively select specific cell types out of heterogeneous cell populations.
In positive selection techniques, labeled antibodies are used to specifically bind the desired cells in the biopsy. The unwanted cells remain unlabeled and are removed. After the selection process, the desired cells have to be detached from the antibodies with a suitable solvent. As a consequence, the stem cells in the transplants are exposed to a medium that may negatively influence cell differentiation and, thus, the transplantation efficacy.
Currently, the majority of stem cell transplants used in animal and human studies aimed at improving neural and vascular tissues have been produced by positive selection of cells carrying specific surface proteins, followed by culturing of the selected cells to increase their numbers. It has, however, never been proven that the positively selected cells indeed have the capacity to differentiate into functional neurons and that they secrete the trophic factors required for full restoration of the neural system.
Generally, the results of intrathecal or intracerebral stem cell transplantations using stem cell transplants obtained by positive selection fluctuate and the beneficial effects are limited. This is most likely due to the fact that it is not known whether the desired beneficial stem cell types are present in the transplant preparations, or that the desired stem cell types lost their therapeutic capability during the timely stem cell processing/culturing process that generally exceeds 72 hours after collection of the bone marrow biopsies.
In negative cell selection, labeled antibodies are used to specifically bind the unwanted cells in the biopsy. The desired cells remain unlabeled and are collected.
Moviglia and coworkers (G. A. Moviglia et al., Cytotherapy 8:196-201, 2006) suggested a cocktail of anti-CD3, anti-CD4, anti-CD19, anti-CD38, anti-CD66b and anti-glycophorin A antibodies to selectively enrich for mesenchymal stem cells as well as anti-CD14, anti-CD16, anti-CD19, anti-CD56 and anti-glycophorin A to enrich CD3-positive cells by immune-rosetting for in vitro experiments.
Patent application U.S. 2002/058289 describes the treatment of bone marrow-derived cell suspensions with a range of antibodies specific for CD2, CD3, CD4, CD5, CD8, CD11b, CD15, CD16, CD19, CD20, CD21, CD22, CD24, CD33, CD38, CD56, CD66b and Glycophorin A to specifically enrich bone marrow biopsies for mesenchymal progenitors.
Patent application WO 02/089726 describes a method for making a homogeneous preparation of hematopoietic stem cells using a combination of cross-flow elutriation and labeling of unwanted cells with magnetically labeled antibodies. The method uses culturing of the resulting enriched cell populations and cell membrane dyes to verify cell identity, which makes the method impractical for therapeutic applications.
U.S. Pat. No. 5,087,570 describes a method for preparing a hematopoietic cell composition using a combination of positive and negative cell selection. The process relies on the use of an antibody to the Sca-1 antigen, which is associated with murine clonogenic bone marrow precursors of thymocytes and progeny T-cells. The Sca-1 antibody is not useful for isolating human hematopoietic cells.
U.S. Pat. No. 5,137,809 describes a method and kit for identifying and analyzing lineages and maturational stages of hematopoietic cells. The method uses a first monoclonal antibody (CD45) labeled with a fluorochrome to react with all leukocytes in a sample, followed by secondary monoclonal antibodies (CD15, CD16, CD10, CD34, CD20, CD19, CD14, CD3, CD11b) labeled with a second fluorochrome to react with a subpopulation of leukocytes. Cell selection is based on flow cytometry.
Stem cell transplants based on negative cell selection, in which cell types of the innate and adaptive immune system (macrophages, lymphocytes, granulocytes and the like) and erythroid cells (erythroblasts, erythrocytes) are removed, have been used in animal and human autologous transplantation studies. The studies using the transplants demonstrated that the beneficial efficacy still remained limited, preventing further clinical application.
Using a mouse model of spinal cord injury, it was confirmed that the stem cell transplants in which the cell types of the immune system and the erythroid cells were removed by negative cell selection have a limited efficacy when transplanted intrathecally.
This is due to the presence of cell types or components with a pro-inflammatory capability in the stem cell transplants, resulting in a negative effect on their therapeutic efficacy.
For these reasons, there is an unmet need for stem cell transplants that lack the pro-inflammatory capability when transplanted to an allogeneic or autologous recipient and that, as a result, will improve the efficacy of stem cell transplantation therapies.